N-glycans on immunoglobulins (Igs) can play a profound and hitherto unappreciated role in their biological function. This study was prompted by the paradoxical observation that isotype-matched monoclonal antibodies (Abs) differed in their capacities to activate the classical complement pathway. A pair of IgG2a Abs had similar monosaccharide composition yet differed in binding to lectins. These properties were lost when the Abs were denatured, suggesting that differences between the native Abs reflected N-glycan orientation rather than the lectin binding capacity of the glycans themselves. Experiments using glycosyltransferases and glycosidases to probe N-glycan accessibility supported this hypothesis. The relative susceptibility to glycosyltransferases and glycosidases and accessibility to lectins was inversely related to the ability of the two Abs to bind C1q, the initial reactive component of the complement system's classical pathway. Deglycosylation of the Ab bearing the more accessible glycan enhanced its capacity to activate complement, suggesting that the more accessible glycan might inhibit C1q binding. In contrast, removal of the other Abs glycan reduced its complement activation potential. There were many sequence differences in the variable domains of the two Abs but the sequence encoding their heavy chain constant regions was identical, suggesting that variable domain sequence affects glycan orientation in the constant region. An isotype-matched pair of human Abs with similar characteristics has also been identified. This proposal seeks to define the influence of Ig variable (V) domains on the orientation of the N-glycan using recombinant and then mutated human and mouse Abs. A detailed structural analysis of glycan structure in relation to function will also be performed. Additional experiments will determine if the observations using the isotype-matched pairs of Abs as an initial model system represent a more general biological phenomenon and determine if glycan-modulated complement activation affects other biological properties of Igs such as clearance of immune complexes from the circulation and deposition in glomeruli. The preliminary observations and proposed studies represent a fundamental paradigm shift in our understanding of Abs activate the complement system and will provide valuable insights into the pathogenesis of autoimmune and other inflammatory diseases as well as in the rational design of recombinant therapeutic Abs.